Kinase activation of ClC-3 accelerates cytoplasmic condensation during mitotic cell rounding

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.     

Function and dynamics of auxin and carbohydrates during earlywood/latewood transition in scots pine

In temperate regions the annual pattern of wood development is characterized by the formation of radially narrow and thick walled latewood cells. This takes place at the later part of the growing season when cambial cell division declines. To gain new insight into the regulation of this process, micro-analytical techniques were used to visualize the distribution of indole-3-acetic acid (IAA), soluble carbohydrates, and activities of sucrose (Suc)-metabolizing enzymes across the cambial region tissues in Scots pine (Pinus sylvestris). The total amount of IAA in the cambial region did not change with latewood initiation. But its radial distribution pattern was altered, resulting in an increased concentration in the cambial meristem and its recent derivatives. Thus, initiation of latewood formation and cessation of cambial cell division is not a consequence of decreased IAA concentrations in dividing and expanding cells. Rather, IAA most likely has a role in defining the altered developmental pattern associated with latewood formation. Carbohydrates and enzyme activities showed distinctive radial distribution patterns. Suc peaked in the phloem and decreased sharply to low levels across the cambial zone, whereas fructose and glucose reached their highest levels in the maturing tracheids. Suc synthase was the dominating Suc cleaving enzyme with a peak in the secondary wall-forming tracheids and in the phloem. Soluble acid invertase peaked in dividing and expanding cells. Suc-phosphate synthase had its highest activities in the phloem. Activities of cell wall bound invertase were low. The absence of major seasonal variations indicates that carbohydrate availability is not a trigger for latewood initiation. However, steep concentration gradients of the sugars suggest a role for sugar signaling in vascular development.     

Indole-3-Acetic Acid Controls Cambial Growth in Scots Pine by Positional Signaling

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development (C. Uggla, T. Moritz, G. Sandberg, B. Sundberg [1996] Proc Natl Acad Sci USA 93: 9282–9286). This idea postulates that the width of the radial concentration gradient of IAA regulates the radial number of dividing cells in the cambial meristem, which is an important component for determining cambial growth rate. The relationship between IAA concentration in the dividing cells and growth rate was poor, although the highest IAA concentration was observed in the fastest-growing cambia. The radial width of the IAA concentration gradient showed a strong correlation with cambial growth rate. The results indicate that IAA gives positional information in plants.     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

白花泡桐不定根发生过程中内源激素和RNA的变化

白花泡桐（Ｐａｕｌｏｗｎｉａｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．）成年型和幼年型茎切段体外培养时不定根发生过程中内源ＩＡＡ、ＣＴＫ和ＡＢＡ含量测定表明：幼年型材料中内源ＩＡＡ和ＣＴＫ的含量在诱导的第２天同时达到高峰,而成年型材料中ＩＡＡ和ＣＴＫ含量的高峰则在第４天出现．两种类型切段根原基出现的时间都与其内源ＩＡＡ和ＣＴＫ的高峰一致．幼年型材料的内源ＡＢＡ含量在第４天达到高峰,随后迅速下降．成年型材料中内源ＡＢＡ则逐步下降．成年型和幼年型材料中ＲＮＡ的变化相同,在诱导的第２天稍有下降,随后显著增加．结果显示,不定根的发生与其内源激素和ＲＮＡ的变化密切相关．     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.     

The influence of cell contact on the division of mouse 8-cell blastomeres

The pattern of division of polarized 8-cell blastomeres with respect to the axis of cell polarity has been compared (i) for cells dividing alone with cells dividing in pairs, and (ii) for early and late dividing cells within a pair. Cell interactions do not seem to influence significantly the overall pattern of division within the population. The only significant difference found was that the second dividing cell in a pair tended to divide in the same way as its earlier dividing companion slightly more frequently than expected. These results suggest that cell interactions immediately prior to and during division do not influence strongly the orientation and position of the division plane. In contrast, interactions between the cells within an intact early 8-cell embryo, which is subsequently disaggregated to singletons or pairs, do influence the type of progeny generated at division to the 16-cell stage, and seem to do so via an effect on the size of the microvillous region generated at the cell apex.     

Relationship between cell division and endogenous auxin in synchronously-cultured tobacco cells

In the cultured tobacco cell, we succeeded in obtaining a partial synchronization of cell division by a combination of pre-starvation, rhythmic light-dark pre-treatment and air tight pre-conditioning. The mitotic index increased during the light period according to the time interval after the end of pre-treatment, and reached its maximum (max=12%) at about 2.5 hr of irradiation, and about 80% of cells completed division 1.5 hr thereafter. During this period, IAA was biosynthesized in the cells, though these cells had been cultured in Murashige and Skoog's medium with 1 mg/l of 2,4-D as a growth substance. IAA was identified by paper chromatography, followed byAvena curvature test and combined gas chromatography-mass spectrometry. The time course of the increase and decrease in the amount of free IAA was parallel to that of the mitotic index. On the other hand, bound IAA increased later and decreased gradually after the end of cell division. Free IAA may have an important role in mitosis.     

Developmental Patterns of Phytohormone Content in the Cortex and Pith of Potato Tubers as Related to Their Growth and Starch Content

The contents of ABA, IAA, and cytokinins (zeatin + zeatin riboside) were determined in the cortex and pith of medium-sized (25 g) and large (120 g) tubers of potato (Solanum tuberosum L., cv. Malakhit) at the end of the flowering phase (48 days after sprouting) and related to plant growth (cell division and enlargement) and starch biosynthesis and deposition. The patterns of phytohormone distribution were different in the cortex and pith. In the latter, with most cells at the enlargement phase, the IAA content increased, as well as the ratios IAA/ABA and IAA/cytokinins. In the cortex dominated by the dividing cells, the ABA content declined, and the ratio cytokinins/ABA exceeded that in the pith. The enhancement of starch synthesis and accumulation in the pith and the retardation of these processes in the cortex followed the changes in the ABA content providing indirect support to the previously made observation that the exogenous ABA promoted starch biosynthesis. The data obtained are discussed in the context of the hormonal control of carbohydrate metabolism and starch deposition in growing tubers.     

Factors Affecting Phytoalexin Production in Cultured Carrot Cells

The production of 6-methoxymellein, a phytoalexin, in culturedcarrot cells under various growth conditions was studied usingtwo induction methods; by adding partial hydrolysates obtainedby treating the cells with pectinase or trypsin, or by directlyadding these enzymes to growing cells to cause the release ofcellular components as endogenous elicitor. 6-Methoxymellein production depended greatly on the cell cultureage. Maximal production was found in cells at the early stationaryphase, while actively dividing cells had only negligible amounts.Release of elicitor from the cells by pectinase or trypsin wasalso influenced by the culture stage. Effective elicitor wasobtained only from cells in the late logarithmic and early stationaryphases. 6-Methoxymellein production required the presence of 2,4-D.IAA could not substitute for 2,4-D, though partial hydrolysatesprepared from these cells grown with IAA or without auxin showedsignificant elicitor activity. On the other hand, the productionwas inhibited by actinomycin D or cycloheximide, suggestingthat de novo syntheses of RNA and protein are required for thephytoalexin production. (Received November 17, 1984; Accepted March 2, 1985)     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

缺磷胁迫对小麦根细胞周期蛋白基因cyc1At表达的影响

用液培方法研究了缺磷胁迫对小麦(TriticumaestivumL.)根系生长的影响。结果表明,随着介质磷水平的提高,小麦根轴长度和植株生长素浓度均降低。在低磷条件下用生长素极性运输抑制剂三碘苯甲酸(TIBA)处理后,小麦的根轴长度明显降低,表明生长素参与了缺磷小麦根轴生长的调控。缺磷小麦根部生长素浓度的提高诱导了细胞周期蛋白基因cyclAt的表达,促进了根分生组织细胞的分裂并驱动了根的生长。     

Demonstrating the localization of hemoproteins by an electron cytochemical method]

The dynamics of division and interphase cell size in the growing ovule of Pinus silvestris was studied on histological slides in the year of fertilization. A constant extension in size of dividing cells of the endosperm was shown which, in this respect, do not differ much from the interphase ones. Cell division and elongation in this tissue occur simultaneously to be completed only with the transition of cell to differentiation. The size extention of the dividing cell does not influence the frequency of cell division. In the integument and the nucleus, the value of dividing cells is relatively constant. At the transition of these cells to elongation their mitotic activity decreases shaply to stop completely after-wards. Cell division and elongation are divided here in time.     

The Effect of Growth Hormones on Cell Division and Expansion in Liquid Suspension Cultures of Acer pseudoplatanus

The effects on cell division and cell size of indole-3-aceticacid (IAA), gibberellic acid (GA), and kinetin were studiedin liquid suspension cultures of cambial cells derived fromAcer pseudoplatanus. It was shown that all three hormones promotecell division and that the effects of both GA and kinetin areadditive to those of IAA, but the effects of GA and kinetintogether are not additive. Treatment with IAA resulted in anincrease of mean cell size (indicating that cell expansion ispromoted), but after GA or kinetin treatment the mean cell sizewas smaller, indicating that little cell expansion had takenplace after each division. The results are discussed in relationto previous work on the effects of hormones in the intact cambiumand to current theories on the interactions of growth hormones.     

Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius. On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced.     

Correlation Between Appearance of Embryogenic Cells and the IAA Levels in Rice Somatic Cell Culture

Changes of endogenous IAA level and IAA action in cultured rice ( Oryza sativa L. ) somatic cells during the period from 7th to 15th day which was the transition from somatic to embryogenic cells were observed. The study was carded out in three experimental systems viz. mature caryopsis and young panicles (2 ~ 5 mm long) of rice cv. "Guangluai 4" under normal osmosis (3% sucrose), mature caryopses from rice cv. "Yanjing 2" or "Guangluai 4" under normal and higher osmosis (5% sucrose or 2.5 % sorbitol). During this period, endogenous IAA contents were greatly increased in young-panicle calli under normal osmosis and mature-caryoptic calli under higher osmosis but decreased in mature-caryoptic calli under normal osmosis. Exogenous IAA could induce the appearance of embryogenic cell from nonembryogenic callus at a lower frequency. And 2,3,5-tri-iodobenzoic acid could increase the frequency of embryogenic cell induction. From these results it could be concluded that accumulation of higher IAA level in the cultured rice cells was essential for induction of embryogenic cell appearance. Since 2,4-D was involved in all induction medium with the same concentration but exerted different effects on embryogenic cell induction, it was suggested that it might act through mediating the endogenous IAA metabolism.     

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division.
The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.     

Oxidative stress and formation and maintenance of root stem cells

The hypothesis of L. Feldman and his coworkers, according to which a more oxidizing environment in the cells of root quiescent center results from high activity of ascorbate oxidase activated by indoleacetic acid (IAA) accumulating in these cells, is discussed. The high activity of ascorbate oxidase is responsible for lowered concentrations of the reduced form of ascorbic acid and glutathione and high content of reactive oxygen species in quiescent center cells. The oxidative stress represses proliferation of the cells. Inhibitors of IAA transport attenuate the oxidative stress, thus suggesting a role of IAA as an activator of ascorbate oxidase. Interestingly, the high concentration of IAA in dividing cap cells adjacent to the quiescent center cells did not cause retardation of cell proliferation and oxidative state in these cells.     

Spatial patterns of cells in dividing epithelia.

The spatial patterns of cell boundaries in a view of the apical surface of a dividing epithelium are explored by constructing a hypothetical cell pattern of an epithelium of dividing cells. The two elements specified in the hypothetical pattern are the orientation of division planes and the separation between the division planes in neighbouring cells. The orientations of division planes in one generation are all the same but are orthogonal to those in the preceding generation. The division-plane orientations follow in an orthogonal succession, as happens in early embryos. The division planes in neighbouring cells are offset. The contractions of division planes that would occur during cytokinesis distort existing boundaries creating various types of cell shapes. The patterns generated resemble cell patterns found in life. The hypothetical pattern is regenerative and shows how epithelial cell patterns where cells divide might arise. It has enabled the putative identification of sister cells and first cousins in the embryonic chick chorion.